(Investigative Ophthalmology and Visual Science. 2000;41:4203-4208.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Interleukin-1 Receptor Antagonist Therapy and Induction of Anterior Chamber–Associated Immune Deviation–Type Tolerance after Corneal Transplantation
Jun Yamada,
Su-Ning Zhu,
J. Wayne Streilein and
M. Reza Dana
From the Laboratory of Immunology, Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
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Abstract
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PURPOSE. Topical treatment with interleukin 1 receptor antagonist (IL-1ra) can
promote corneal allograft survival by suppressing induction of
allodestructive immunity. The purpose of these experiments was to
determine whether IL-1ra could also promote induction of
allo-protective tolerogenic pathways, including anterior
chamber–associated immune deviation (ACAID), which has been shown to
participate in long-term survival of corneal transplants.
METHODS. Corneal buttons from BALB/c (syngeneic) or C57BL/6 (fully mismatched
allogeneic) mice were orthotopically grafted onto BALB/c recipients.
Topical IL-1ra or vehicle alone was applied to grafts three times
daily. Donor-specific ACAID was measured in allogeneic grafted mice at
4 and 8 weeks after transplantation by ear-challenging grafted hosts
with donor-derived splenocytes 1 week after SC immunization. In
separate experiments, grafted mice were treated for 4 weeks before
injecting ovalbumin (OVA) into their anterior chambers to determine
their capacity to induce antigen-specific ACAID.
RESULTS. Treatment with IL-1ra did not promote, or inhibit, induction of
donor-specific ACAID compared with vehicle-treated controls at either
the early or late time points studied. However, IL-1ra treatment after
transplantation led to significantly earlier restoration of the grafted
eyes’ capacity for inducing ACAID to soluble antigen (OVA).
CONCLUSIONS. Promotion of OVA-specific ACAID by IL-1ra suggests that suppression of
IL-1–mediated mechanisms contributes to recovery of the anterior
segment’s immunosuppressive microenvironment at least 1 month earlier
than would otherwise be seen after corneal transplantation. However,
IL-1ra treatment does not alter induction of donor-specific ACAID after
transplantation, suggesting that its anti-inflammatory activities do
not lead to an ACAID-inducing signal per se. This suggests that IL-1ra
promotes graft survival almost exclusively by virtue of suppressing
inflammation and not by directly promoting tolerance or
antigen-specific regulatory pathways.
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Introduction
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Corneal transplantation has emerged as the most common and
successful form of tissue transplantation. The extraordinary success of
corneal transplants has been related to its immune privileged status in
the ocular microenvironment,1
where orthotopic corneal
allografts can experience prolonged survival even without
immunosuppressive treatment.2
3
This is all the more
remarkable given that corneal graft recipients universally become
sensitized to donor-derived antigens after transplantation regardless
of the final outcome of the transplanted tissue.3
4
However, in spite of this universal allosensitization, it still remains
unknown why some grafts get rejected and others survive indefinitely.
It has been proposed that this may be related to acquisition of
tolerance by some hosts, as evidenced by the observation in laboratory
animals that long-term acceptance of corneal grafts is associated with
a form of donor-specific tolerance known as allospecific anterior
chamber–associated immune deviation (ACAID).3
Moreover,
induction of donor-specific ACAID has been shown to promote graft
survival, suggesting that induction of this form of allospecific
tolerance may play a critical role in long-term corneal transplant
survival.5
Interestingly, the time course for induction of alloreactive responses
in experimental corneal transplantation does not perfectly coincide
with that for induction of tolerogenic signals—a fact that may explain
the observation that the preponderance of corneal graft rejections
(whether in humans or rodents) occur in the relatively early period
after transplantation. For example, in the mouse model, although
induction of allodestructive delayed-type hypersensitivity (DTH) occurs
very early after transplantation, induction of tolerogenic
ACAID-promoting signals takes at least 8 weeks after orthotopic
grafting.3
The exact reason for the observed delay in
induction of tolerance remains unknown, but it has been demonstrated
that the procedure of transplantation itself (even in the syngeneic
setting) is sufficient to perturb the ocular microenvironment and
abrogate the eye’s, or aqueous humor’s, normal capacity to support
ACAID induction for several months.6
7
It has been
postulated that surgical manipulation of eyes may induce the
overexpression of a wide array of inflammatory cytokines including
interleukin (IL)-1, which potentiates immune responsiveness and thereby
suppresses ACAID induction.6
8
IL-1 has a wide range of activities that promote immunoinflammatory
responses, including critical mediation of the acute-phase response,
chemotaxis, activation of inflammatory and antigen-presenting cells,
upregulation of adhesion and costimulatory factors on cells, and
stimulation of neovascularization.9
IL-1 receptor
antagonist (IL-1ra) is a naturally occurring IL-1 isoform with
high-affinity binding to IL-1 receptors but with no agonistic activity
even at high concentrations, and hence is capable of profound
downregulation of IL-1–mediated responses in both humans and
rodents.9
There are at least two separate, not necessarily
mutually exclusive, mechanisms that can explain IL-1ra’s suppression
of alloimmunity and graft rejection: (a) active suppression of
sensitization (i.e., priming of allodestructive Th1 cells), and/or (b)
promotion of tolerance to donor antigens. Because topical IL-1ra can
prevent DTH-type sensitization to corneal grafts by downmodulating
antigen-presenting cell function and local
inflammation,4
8
we performed the experiments described
herein to determine whether topical treatment with IL-1ra can similarly
promote the early induction of tolerogenic ACAID in grafted eyes. Two
models were studied1
: recovery of the grafted eye’s
immune privileged microenvironment and capacity to support induction of
ACAID to intracamerally injected soluble antigen, and2
the
rapidity with which the host can acquire donor-specific ACAID.
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Materials and Methods
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Mice
Eight- to 10-week-old male mice were purchased (Taconic, NY).
Animals with dystrophic/degenerative corneal calcific deposits were
excluded from study. All animals were treated according to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
BALB/c (H-2d) strain mice were used as recipients
and C57BL/6 (MHC and minor alloantigen disparate,
H-2b) or BALB/c (syngeneic) strain mice as
donors. C3H/HeN mice were used as third-party controls for specificity
of the immune response.
Orthotopic Corneal Transplantation
Our usual method for corneal transplantation has been described in
detail elsewhere.3
4
10
Briefly, all animals were deeply
anesthetized before surgical procedures. The central 2-mm of the donor
cornea was excised and secured in recipient graft beds with eight
interrupted 11-0 nylon sutures (Sharpoint; Vanguard, Houston, TX).
Antibiotic ointment was applied to the corneal surface, and the lids
were closed for 24 hours with an 8-0 nylon tarsorrhaphy. All grafted
eyes were examined after 72 hours; grafts with technical difficulties
(hyphema, infection, or loss of anterior chamber) were excluded from
further consideration. Transplant sutures were removed in all cases on
day 7.
Pharmacological Strategy and Assessment of Donor-Specific ACAID
Induction
Topical preparations were applied to recipient mice three times
daily from the day of grafting until the appropriate time point as
detailed below. The study medication was composed of 2% human
recombinant IL-1ra in 0.2% sodium hyaluronate in PBS (Amgen, Thousand
Oaks, CA). Controls received the vehicle 0.2% sodium hyaluronate only.
Allografted, positive, and ACAID control mice were immunized by SC
injection of 10 x 106 C57BL/6 splenocytes.
ACAID control mice in addition received anterior chamber injection of
5 x 105 C57BL/6 spleen cells 1 week before
SC immunization.7
Neither the positive or ACAID controls
were grafted. Seven days after immunization, 1 x
106 irradiated (2000 rad) C57BL/6
splenocytes in 10 µl Hanks’ balanced salt solution (HBSS) were
injected into the right ear pinnae. At 24 and 48 hours after ear
challenge, ear thickness was measured with a low-pressure micrometer
(Mitutoyo, MTI Corporation, Paramus, NJ). Ear swelling was expressed as
follows: specific ear swelling = (24-hour measurement of right
ear - 0-hour measurement of right ear) - (24-hour
measurement of left ear - 0-hour measurement of left ear) x
10-3 mm. Ear swelling responses at 24 hours
after injection are presented as a group mean ± SEM measurement.
Assay for the Ability of an Eye to Support ACAID Induction
Animals received 50 µg ovalbumin (OVA) in 3 µl HBSS into the
anterior chamber of syngeneically grafted eyes. Anterior chamber
inoculations of ungrafted normal eyes served as ACAID controls. Seven
days later these animals and positive controls (not receiving
intracameral antigen) were immunized with OVA (100 µg) emulsified 1:1
in CFA in a total volume of 100 µl injected SC into the nape of the
neck. In antigen-specificity control experiments some animals were
immunized with bovine serum albumin (BSA) emulsified in CFA. Seven days
after SC immunization, mice received intradermal inoculation of OVA or
BSA (200 µg/10 µl of HBSS) into the right ear pinnae, and the
antigen-specific ear swelling response was assessed as described above.
For all experiments, DTH responses after antigenic challenge that were
significantly lower than that observed in positive controls denoted
ACAID.
Statistical Methods
Statistical analyses were performed by using the Student’s
t-test for comparison of DTH responses. Second, we
constructed Kaplan–Meier survival curves and used the
Breslow–Gehan–Wilcoxon test to compare the probability of corneal
graft survival. All P values < 0.05 were deemed
significant.
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Results
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Induction of Allospecific ACAID after Corneal Transplantation
As reported previously,10
IL-1ra treatment
(n = 15) led to significant reduction in the rate of
allograft rejection (Fig. 1A ) compared with controls (n = 10) treated with vehicle
alone (P = 0.03). Because we have previously shown that
IL-1ra can suppress induction of donor-specific DTH,4
in
the current experiments we aimed to determine whether IL-1ra could
similarly affect the induction of tolerogenic mechanisms such
allospecific ACAID, which is normally observed 8 weeks after
transplantation in accepted grafts.3
At the 8-week time
point, all mice, except for negative controls, were SC immunized with
donor splenocytes and subsequently challenged with donor cells to assay
for donor-specific tolerance (Fig. 1B)
. Except for one host with an
accepted graft demonstrating heightened alloreactivity, the data
indicate that the acceptors, regardless of their treatment regimen,
exhibited suppressed allospecific responses compared with rejectors or
positive controls (P < 0.001). There was a
preponderance of grafted hosts treated with IL-1ra demonstrating
allospecific ACAID, but the direct association observed was between
graft acceptance and ACAID and not between IL-1ra treatment per se and
ACAID induction, as reflected by the hosts treated with IL-1ra that
failed to exhibit donor-specific tolerance.
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Figure 1. Corneal graft survival (A) and induction of donor-specific
ACAID (B) in mice treated with topical IL-1ra or vehicle for
8 weeks. Topical therapy with IL-1ra leads to significant promotion of
allograft survival (P = 0.03) as demonstrated by
Kaplan–Meier analysis (A). Donor-specific responses were
measured after allospecific challenge in the ears of negative (Naive)
controls, positive controls sensitized to donor splenocytes (Primed),
and allografted mice either treated with IL-1ra (IL-1ra Tx) or vehicle
alone (Vehicle Tx). Mean 24-hour antigen-specific DTH ear swelling
responses show that except for one host, long-term corneal graft
acceptors, regardless of treatment regimen, exhibit donor-specific
tolerance compared with primed positive controls; *P <
0.001. Similar results were obtained at 48 hours.
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Capacity of Grafted Eyes to Mount Antigen-Specific ACAID to
Intracameral Soluble Antigens
Previous studies have shown that the capacity of the eye to
support "deviant" or tolerizing immunity to intracamerally injected
antigens is lost for at least the first 8 weeks after transplantation,
even in the syngeneic setting.6
To test whether the
anti-inflammatory properties of IL-1ra could restore this capacity
earlier than would otherwise be possible, the capacity of the grafted
eye to induce ACAID to a nominal antigen (OVA) was tested under cover
of IL-1ra treatment. Four weeks after syngeneic corneal grafting, OVA
was injected into the anterior chamber of grafted eyes treated since
the time of transplantation with IL-1ra (n = 5) or with
vehicle alone (n = 5). ACAID control mice were those
receiving intracameral injection of OVA into ungrafted eyes. All mice,
including positive controls that had not received intracameral antigen,
were subsequently immunized with OVA in adjuvant and ear-challenged
(Fig. 2)
. Injection of OVA into eyes bearing corneal grafts and treated with
vehicle alone universally failed to prevent OVA-specific DTH. By
contrast, injection of OVA into all grafted eyes treated with IL-1ra
led to impaired development of OVA-specific response (P = 0.008). These findings suggest that the microenvironment of the
grafted eye treated with IL-1ra can support induction of ACAID to
soluble antigens significantly earlier than would otherwise be possible
in untreated eyes.
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Figure 2. ACAID induction to intracameral ovalbumin (OVA) 4 weeks after syngeneic
corneal transplantation. Grafted eyes were treated with either IL-1ra
or vehicle alone for 4 weeks at which point 50 µg OVA was injected
intracamerally into grafted eyes. All the grafted eyes and negative
(Naive) controls, positive controls SC immunized to OVA (Primed), and
ungrafted animals receiving intracameral OVA (ACAID controls) were then
ear-challenged by OVA. Mean 24-hour ear swelling responses (comparable
results at 48 hours) show that grafted eyes treated with IL-1ra have
restored their capacity to induce OVA-specific ACAID compared with
vehicle-treated or positive controls; *P = 0.008.
All tests were repeated at least once.
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Possibility of Early Induction of Allospecific ACAID after
Transplantation
Because data from the OVA experiments suggested that topical
application of IL-1ra could promote swift recovery of the
ACAID-supporting microenvironment of the eye, we embarked on a series
of experiments to test whether IL-1ra could likewise induce ACAID to
donor antigens in the early postoperative period because this could
provide one possible mechanism to explain this cytokine’s promotion of
corneal graft survival. Three weeks after allotransplantation, vehicle
(n = 6) or IL-1ra–treated (n = 6)
grafted (and control ungrafted BALB/c; n = 5) mice were
immunized with C57BL/6 splenocytes before ear challenge at 4 weeks
(Fig. 3)
. The data suggest that treatment with IL-1ra does not lead to early
induction of allospecific ACAID. Rather, the data clearly show that SC
immunization of the host to donor antigens, early after
transplantation, leads to a robust allospecific response regardless of
the treatment rendered during the engraftment period.
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Figure 3. Donor-specific ACAID 4 weeks after corneal allotransplantation. Grafted
mice were treated with either IL-1ra or vehicle alone. ACAID controls
were ungrafted but received intracameral injection of donor splenocytes
1 week before immunization. Mean 24-hour ear swelling responses
(comparable results at 48 hours) demonstrate that IL-1ra-treated hosts,
in contrast to ungrafted ACAID controls, are not capable of exhibiting
donor-specific tolerance; *P < 0.0001. All tests
were repeated at least once.
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Necessity of Continuous Treatment with IL-1ra for Promoting Graft
Survival
The data above suggest that although IL-1ra can restore the ocular
microenvironment sufficiently to induce ACAID to soluble antigen, it is
incapable of early induction of allospecific ACAID. However, because
IL-1ra does not bias the host response against generation of tolerance
as the 8-week data show (Fig. 1B) , we hypothesized that a short course
of therapy could still promote long-term graft survival, by suppressing
allospecific DTH early after surgery and still allowing for the normal
induction of ACAID at later time points. To test for this possibility,
hosts of allografts received IL-1ra (n = 15) or vehicle
(n = 13) only for 4 weeks after grafting. All treatment
was terminated thereafter, and animals were followed for signs of
rejection (Fig. 4A
). Subsequent to completion of treatment, grafts treated previously
with IL-1ra retained clarity for another 2 weeks and exhibited lower
rejection rates compared with grafts treated with vehicle alone
(P < 0.05 at day 45). However, 5 (33.3%) of 15 grafts
on early IL-1ra treatment were subsequently rejected, mostly after
termination of treatment, so that by 8 weeks there was no statistical
difference between the two treatment groups (P =
0.062). To ensure that early treatment with IL-1ra would not prejudice
late induction of allospecific ACAID after termination of treatment,
all mice (except for naive negative controls) were immunized with donor
splenocytes before ear challenge (Fig. 4B)
. With the exception of one
host that demonstrated significant alloreactivity, acceptors
demonstrated donor-specific ACAID, and conversely, all rejectors
exhibited strong allospecific responses regardless of their previous
treatment regimens (P < 0.05). Therefore, although
IL-1ra does not bias against the generation of ACAID induction,
long-term therapy is indicated to prevent host sensitization and graft
rejection.
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Figure 4. Eight-week corneal allograft survival (A) and donor-specific
ACAID (B) in animals treated with IL-1ra or vehicle for 4
weeks. Kaplan–Meier analysis reveals that although early cessation of
IL-1ra therapy can maintain graft survival for a short period, the
rejection rate increases thereafter leading to an overall 8-week
survival rate not appreciably different from that among vehicle-treated
controls (A). Mean 24-hour antigen-specific responses show
that only acceptors exhibit tolerance, regardless of treatment with
IL-1ra; P < 0.05.
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Antigen and Site Specificity of IL-1ra Modulation of Immune
Responses to Ocular Antigens
Three separate series of experiments were performed to test
whether the modulation of immunity to ocular antigens as a result of
IL-1ra treatment is antigen-specific and limited to antigens introduced
to the specific eye thus treated. In the first series of experiments we
wanted to determine whether the observed suppression of DTH reactivity
to graft antigens as a result of IL-1ra therapy also extended to
unrelated (third-party) antigens (Fig. 5A
). BALB/c hosts (n = 12) were grafted with C57BL/6
corneas and were randomized to receive either IL-1ra or vehicle for 3
weeks, after which the grafted eyes were enucleated. One week later
animals were immunized to third-party C3H/HeN antigens by SC injection,
followed 1 week later by ear challenge to C3H/HeN antigens. Positive
controls (n = 4) were treated with vehicle only and
were immunized to C3H/HeN antigens before ear challenge; negative
controls (n = 4) were treated with IL-1ra and were not
immunized before ear challenge. Our results showed that ocular
treatment with IL-1ra did not suppress generation of C3H-specific DTH
(Fig. 5A) .
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Figure 5. The effect of ocular application of IL-1ra is tested on irrelevant
transplantation (A) and soluble antigens (B).
BALB/c mice received allogeneic orthotopic grafts (n =
12) from C57BL/6 donors (A). Allografted eyes received
IL-1ra before immunization of hosts with C3H/HeN antigens and ear
challenge with irradiated C3H/HeN splenocytes (Exp group). Positive
controls (n = 4) were treated with vehicle only and
were immunized to C3H antigens before ear challenge; negative controls
(n = 4) were treated with IL-1ra and were not immunized
before ear challenge. Mean 24-hour ear swelling responses show that
ocular treatment with IL-1ra does not suppress generation of
C3H-specific immunity; *P < 0.001 compared with
negative controls. (B) BALB/c animals (n =
12) were syngeneically grafted and treated with IL-1ra before
intracameral injection with OVA. One week later they were immunized to
BSA followed by ear challenge to BSA (Exp group). Positive controls
(n = 4) did not receive any intracameral antigen but
were immunized to BSA before ear challenge; negative controls
(n = 4) did not receive either intracameral antigen or
SC immunization before ear challenge. Mean 24-hour data show that there
is normal induction of BSA-specific DTH; *P < 0.001
compared with negative controls. Similar results were obtained at 48
hours. Hence, downmodulation of DTH to ocular antigens as a result of
IL-1ra therapy is antigen-specific.
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In a second set of experiments we determined whether the
ACAID-promoting effect of IL-1ra on grafted eyes also extended to
irrelevant antigens. BALB/c animals (n = 12) were
syngeneically grafted and treated with IL-1ra for 4 weeks at which
point the grafted eyes were intracamerally injected with OVA. One week
later they were immunized SC to an unrelated antigen, BSA, followed by
ear challenge to BSA. Positive controls (n = 4) did not
receive any intracameral antigen but were immunized to BSA before ear
challenge; negative controls (n = 4) did not receive
either intracameral antigen or SC immunization before ear challenge. As
summarized in Figure 5B
, the generation of ACAID response to
intracameral OVA that we had observed in grafted eyes treated with
IL-1ra (Fig. 2)
did not extend to an irrelevant antigen BSA, suggesting
that suppression of Th1-type immunity to ocular antigens as a result of
treatment with IL-1ra did not extend to antigens presented to the host
at nonocular sites. Finally, in another set of experiments we
determined whether application of IL-1ra to one eye can modulate the
immune response to antigens presented to the fellow eye. Syngeneic
grafts were performed in the right eyes of animals (n =
12), whereas their unmanipulated left eyes were treated with IL-1ra for
4 weeks. This was followed by SC immunization with OVA in CFA and ear
challenge 1 week later. Negative controls (n = 4)
received IL-1ra but were not SC immunized before ear challenge. There
was no sign of OVA-specific ACAID in animals that received OVA in their
grafted (right) eye but had received IL-1ra in their fellow (left) eye;
mean (± SEM) 24-hour swelling response was 83 ± 7 µm compared
with 25 ± 6 µm in negative controls (P <
0.001). We concluded from these experiments that the effect of IL-1ra
in modulating the immune response is limited only to ocular antigens in
the eye being treated by it.
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Discussion
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Allospecific unresponsiveness in transplantation can be a
manifestation of either downmodulation in the induction of
alloreactivity (e.g., donor-specific DTH) or active generation of
tolerogenic mechanisms (e.g., allospecific ACAID). As such, the
profound positive effect of IL-1ra on corneal graft survival could
theoretically be a manifestation of either suppressed sensitization or
promotion of regulatory signals—or both. Because induction of ACAID to
ocularly delivered antigens is thought to be an important facet of
ocular immune privilege1
6
and because the generation of
ACAID to alloantigens is believed to play an important role in
tolerizing the host and maintaining long-term acceptance of corneal
grafts,3
5
it is important to determine whether changes to
the ocular microenvironment as a result of cytokine alterations can
modify the eye’s capacity to support this form of deviant immunity.
The experiments described in this report were conducted to test the
effect of IL-1ra treatment of corneal grafts on induction of ACAID-type
tolerance to soluble and donor-derived antigens. The control
experiments that have been performed (Fig. 5)
, evaluating the effect of
IL-1ra on irrelevant or third-party antigens presented to the host at
nonocular sites or to untreated eyes clearly demonstrate that the
effect of topical IL-1ra on modulating immunity to ocular antigens is
both antigen- and site-specific. Our results suggest that although
IL-1ra treatment can promote the restoration of the eye’s capacity to
support induction of ACAID to intracamerally delivered soluble antigen
at least 1 month earlier than would otherwise occur after corneal
transplantation (Fig. 2)
, it does not promote (Fig. 3)
or abolish (Fig. 1B)
the grafted eye’s capacity to induce allospecific tolerance.
Hence, the greater propensity for donor-specific tolerance seen in
IL-1ra–treated animals, when evaluated as a group, appears to be more
a function of the increased rate of acceptance in hosts treated with
this cytokine than a function of the active generation of tolerogenic
signals per se. This conclusion is further supported by our results
demonstrating that early termination of IL-1ra therapy leads to
subsequent graft failure (Fig. 4)
, because induction of tolerance
should have a more long-lasting effect on transplant survival.
It is important to discuss the limitations of our study. Although
we8
and others11
have shown that ocular
antigen presentation and priming of DTH responses can be potentiated by
IL-1, the evidence presented in this report suggests that IL-1ra does
not suppress or promote induction of allospecific ACAID. Our data do
not address the reasons why early restoration of the ocular
microenvironment’s capacity to induce ACAID to soluble antigens cannot
be replicated for alloantigens. It is unlikely that IL-1ra’s failure
to generate early allospecific tolerance is dose-dependent. We have
already established that significantly lower doses of topical IL-1ra
than that used in this study can have comparable efficacy in
suppressing anterior segment inflammation.12
Hence, it is
unlikely that higher doses of IL-1ra can promote early allospecific
ACAID. However, several other possibilities exist that may explain
IL-1ra’s failure to directly generate donor-specific tolerance,
including different mechanisms that may regulate immunity to soluble
compared with alloderived antigens. For example, it is known that
generation of a deviant form of immunity to intraocular antigens is
critically dependent on the functional presence of a camerosplenic axis
responsible for presentation of antigen by APCs of the iris and ciliary
body in the context of immunomodulatory factors (e.g., TGF-ß2, IL-10)
that allow for generation of regulatory cells in the
spleen.13
Although it has been proposed that tolerance to
graft antigens may also involve generation of regulatory cells in the
spleen,1
3
5
7
it appears that corneal alloantigen
processing is largely mediated by corneal and ocular surface dendritic
(e.g., Langerhans) cells.4
8
10
In addition, fundamental
differences in the nature of soluble compared with cell-associated
transplantation antigens, which can affect how these antigens are
processed and presented,14
may affect the capacity of
IL-1ra to modulate the immune response generated to a specific antigen.
Finally, it is possible that allotransplantation leads to more
inflammation than syngeneic transplantation; hence, the same
anti-inflammatory effect of IL-1ra that can restore ACAID to soluble
antigens in the syngeneically grafted eyes may fail to do so in the
allografted eyes. Theoretically, this could be tested by evaluating
induction of ACAID to soluble antigens in allogeneically grafted eyes.
Practically, however, it is not feasible to use an allografted eye to
test for ACAID to an irrelevant antigen (e.g., OVA); allograft
rejection would confound the data derived from the OVA experiments
because we know that rejecting eyes cannot support ACAID because of the
significant inflammation generated during rejection. Moreover,
other data from our laboratory do not support the hypothesis that the
failure of IL-1ra to directly induce allospecific is due to increased
inflammation in the allograft setting. IL-1ra–mediated restoration of
ACAID-inducing ability to soluble antigens has been documented even in
highly inflamed eyes with corneal neovascularization.8
Second, even in the absence of strong allogeneic DTH-inducing
inflammation the acquisition of donor-specific ACAID takes a long
time.7
Hence, it may be that the shedding of antigen from
corneal grafts and their processing by intraocular ACAID-generating
APCs takes a long time regardless of the cytokine milieu of the
anterior segment.
Our data suggest that IL-1ra’s promotion of graft acceptance is
largely by virtue of suppressing sensitization and not by promoting
antigen-specific regulatory pathways per se. Because in the first month
after transplantation IL-1ra can suppress generation of allospecific
DTH, and yet generation of donor-specific ACAID (with or without
IL-1ra) requires at least 2 months, our data would suggest that an
effective way of maximally promoting graft survival with this cytokine
short of its indefinite use is to use IL-1ra sufficiently long to allow
for the normal generation of allospecific tolerogenic signals. In fact,
we have data (not shown here) to support this prediction. Long-term
(>16 weeks) data from our laboratory on animals treated for 8 weeks
with IL-1ra and then followed without any treatment reveal that
allograft rejection beyond 8 weeks (the standard follow-up period) is
in fact rare. From a clinical application standpoint, the failure of
IL-1ra to promote allospecific tolerance would mean that coverage with
IL-1ra for the first several months after surgery may be required to
prevent most cases of graft rejection.
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Footnotes
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Supported in part by National Institutes of Health Grants EY0363 and EY12963 (MRD) and EY19765, EY05678, and AR44130 (JWS); Eye Bank Association of America, Research to Prevent Blindness, Fight-for-Sight, and Amgen (MRD).
Submitted for publication November 5, 1999; revised March 30 and August 16, 2000; accepted August 25, 2000.
Commercial relationships policy: P (MRD).
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1999.
Corresponding author: M. Reza Dana, Laboratory of Immunology, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. dana{at}vision.eri.harvard.edu
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Abstract
Introduction
